Intradermal hpv peptide vaccination

ABSTRACT

The invention relates to the use of a peptide derived from HPV-E2, -E6 and/or E7 protein for the manufacture of a medicament for the treatment or prevention of an HPV related disease, wherein the medicament is for interdermal administration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is Continuation application of U.S. patent applicationSer. No. 12/451,983, filed Apr. 12, 2010, which is the National Phase ofInternational Patent Application No. PCT/NL2008/050315, filed May 27,2008, published on Dec. 31, 2008 as WO 2009/002159 A1, which claimspriority to European Patent Application No. 07109287.8, filed May 31,2007 and U.S. Provisional Application No. 60/941,070, filed May 31,2007. The contents of these applications are herein incorporated byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 15, 2016, isnamed 069818-0391SequenceListing.txt and is 20 KB.

FIELD OF THE INVENTION

The present invention relates to the field of medicine and immunology.In particular it relates to intradermal HPV peptide vaccination.

BACKGROUND OF THE INVENTION

HPV infection is highly prevalent among young, sexually active male andfemale individuals. Large prospective studies showed that acquisition ofHPV from male partners is common, occurring in 40-60% of subjects duringa 3 year follow-up period (Koutsky etal., 1997, Ho etal., 1998, Marrazzoetal., 2000). Therefore, HPV is probably the most common sexuallytransmitted disease.

Papillomaviruses of the high-risk types (e. g. HPV16, 18, 31, 33, and45) are responsible for cervical cancer (Bosch et al., 1995, Zur Hausen,1996). Following

infection of the basal epithelial cells, the immediate HPV early genesE1, E2, E5, E6 and E7 are expressed. The E1 and E2 genes regulate viralreplication. Furthermore, the E2 protein controls the expression of theE6 and E7 oncoproteins. The E6 protein of the high-risk HPV typesspecifically binds to p53 and targets its rapid degradation through theubiquitin pathway. P53 is involved in initiation of apoptosis and lossof this protein result in the prevention of apoptosis (Scheffner etal.,1990). The E7 protein of high-risk types binds to pRB, which normallyprevents cells from entering the cell cycle by inactivating E2F, aprotein needed for cell cycle entry (Dyson et al. 1989). E7 expressionresults in the failure of infected cells to withdraw from the cell cycleand differentiate.

Prolonged and elevated expression of the E6 and E7 oncoproteins istightly associated with HPV-induced dysplasia and transformation intocervical carcinoma.

The protective role of the immune system in the defense againstHPV-related diseases and HPV-induced cancer in humans is suggested bythe fact that compared to normal controls, immunosuppressed renaltransplant patients and patients infected withHIV display a 17-fold greater incidence of genital HPV infection (Hoetal., 1994, Matorras et al. 1991, Halpert et al. 1986). The diminishedcapacity of immunosuppressed individuals to resolve HPV infectionindirectly points towards a protective role of the immune system earlyin infection. Evidence of protection against HPV via immunity againstthe early antigens E2, E6 and E7 comes from the cottontail rabbitpapillomavirus model which is the major animal model forcancer-associated papillomaviruses. Vaccination with the nonstructuralproteins E1 and E2 induces the regression of virus-induced papillomaswhereas viral tumor growth is suppressed.

Furthermore, rabbits vaccinated with the combination of the E 1, E2, E6,and E7 genes were completely protected against viral challenge (Han etal. 1999, Selvakumar et al. 1995). Importantly, rabbits withprogressively growing papilloma virus-induced warts could eradicatetheir lesions as well as clear latent viral infections following twoinjections with a vaccine comprising E6 and E7 long overlapping peptides(Vambutas, Vaccine 2005). These data indicate that immunity against E2,E6, and E7 can be effective as immunoprophylaxis of papillomavirusinfection as well as therapeutically for HPV induced lesions and cancer.

Considerable interest exists in the identification of epitopes involvedin the immune response to HPV16, given the possibility to incorporatethese as subunits into vaccine or to use these epitopes to monitorvaccine induced immunity in vivo. Since most epithelial cells expressMHC class I but not class II, the attention has so far been focused onthe induction of tumoricidal HPV-specific CDS+ cytotoxic T lymphocytes(Melief et al., 2000; Ressing et al., 1995; Ressing et al., 2000;Ressing et al., 1996). HPV specific CDS+ T-cell reactivity has beenfound in the peripheral blood of patients diagnosed with cervicalintraepithelial neoplasia grade III (CIN III) lesions or cervicalcarcinoma (Nimako et al., 1997; Ressing et al., 1996) and intumor-infiltrating T-cell populations isolated from patients withcervical cancer (Evans etal., 1997). Tumor specific CD4+ T helper (“Th”)immunity is now also considered pivotal for the efficient eradication ofsolid tumors, despite the fact that most of these tumors do not expressMHC class II (reviewed in Melief et al., 2000; Pardoll and Topalian,199S; Toes et al., 1999). Recent evidence indicates that CD4+ tumorspecific T-cells are required not only for optimal induction of CDS+tumor specific CTL but also for optimal exertion of local effector cellfunction by these CTL (Ossendorp etal., 199S, Toes etal., 1999). Forinduction of MHC class I restricted tumor-specific immunity,cross-presentation of antigens that have been captured by professionalantigen presenting cells appears to play a dominant role. For properinduction of an effective tum or -specific CTL by cross-primingtumor-specific CD4+ T cell help is required (Toes et al., 1999,Schoenberger et al., 1998).

Strong indications for a protective role of HPV-specific Th-immunity wassuggested by the predomination of CD4+ T-cells in regressing genitalwarts (Coleman et al., 1994) as well as by the detection of delayed-typehypersensitivity responses to HPV16 E7 in the majority of subjects withspontaneous regressing CIN lesions (Hopfl et al., 2000). Furthermore, inmost healthy persons the immune system succeeds in eliminating the virusbefore malignancies develop (Koutsky, 1997; Evander, 1995). In line withthis, more than half of all healthy females tested display strongproliferative HPV16 E2- and E6-specific Th1/Th2 cell memory responses(de Jong, 2002; Welters, 2003; de Jong, 2004). Furthermore,Th-reactivity against E2 was found to occur at time of viral clearance(Bontkes, 1999). Healthy subjects display HPV1 6 E7-specific immunitytoo (Welters, 2003; van der Burg, 2001). In contrast, the occurrence ofHPV-induced cancer is strongly associated with immune failure. Analysisof HPV16 E2-, E6- and E7-specific CD4+ T-cell immunity in the peripheralblood of patients with HPV16+ induced neoplasia revealed that half ofthe patients with high grade vulvar neoplasia (van Poelgeest, 2005) andthe majority of patients with CIN III failed to mount a proper immuneresponse (de Jong, 2004). Of the cervical carcinoma patients tested,approximately half lacked any detectable proliferative T-cell responses.The other half displayed weak proliferative HPV 16 E2- and E6-specificT-cell responses not associated with the production of Th 1/Th2cytokines but with IL-10 (de Jong, 2004). This corroborates previousobservations that E6- and E7-specific proliferative responses can bepresent (Luxton, 2003), but that the peripheral Th1 response in cervicalcarcinoma patients is low (de Gruijl, 1996; de Gruijl, 1998) or lacking(Tsukui, 1996). Because, the CD4⁺ T-cell response is of pivotalimportance for the induction and maintenance of CD8⁺ CTL immunity(Melief, 2002), these data offer a plausible explanation why peripheralHPV 16-specific CTL are rarely detected in patients with high-gradedysplasia or cancer (Ressing, 1996; Bontkes, 2000; Nimako, 1997; Youde,2000), while such CTL are more commonly detectable in women without HPV16+ neoplasia (Nakagawa, 1997; Nakagawa, 1999).

For a clinically relevant approach of immunizing subjects against HPVinparticular, it is preferred that both specific T-helper cells and CTLare induced. We have already shown that immunization with minimal CTLepitopes results in protection against tumors in some models (Kast etal. 1991) whereas, in other models, it can lead to tolerance orfunctional deletion of virus- and tumor-specific CTL that when otherwiseinduced are protective (Toes et al. 1996ab). The occurrence of toleranceor functional deletion decreases the effects of vaccinationsignificantly. Epitopes involved with this effect were therefore notsuitable for immunization purposes. Processing of exogenous antigens forpresentation by MHC class I molecules by cross-priming as well as byother mechanisms is now widely recognized second pathway of processingfor presentation by MHC class I, next to the well known endogenous route(Jondalet al. 1996, Reimann et al. 1997). The normal outcome of antigenprocessing via this pathway is CTL tolerance, unless APC activation byCD4⁺ T-cells takes place (Kurts etal., 1997). To solve this problem oftolerance or functional deletion, WO 02/070006 disclosed the use of longHPV peptides as a vaccine, said peptide having both a MHC Class I and IIpresentable epitopes resulting in the activation of both CD4⁺ and CD8⁺T-cells.

HPV vaccines developed in WO 02/070006 may still further be improvedsince high doses of peptides and/or sequential vaccinations are usuallyused in order to get an optimal immunogenic effect. Furthermore,adjuvants such as Montanide ISA-51 are usually required to get anoptimal immunogenic effect. These adjuvants induce undesiredside-effects such as prolonged local swelling at the site of injection,red swollen hands, fever, vomiting, joint pain, a general illnessfeeling similar to symptoms experienced during influenza infection.These side effects are generally experienced as uncomfortable and willprevent treatment of patients with early stage lesions.

Therefore, there is still a need for improved HPV vaccines, which do nothave all the drawbacks of the existing vaccines, among other, the HPVvaccine used in the invention does not necessitate high doses ofpeptides and/or sequential vaccinations and/or an adjuvant.

DESCRIPTION OF THE INVENTION

The invention relates to the use of a peptide derived from HPV-E2, -E6and/or -E7 protein for the manufacture of a medicament for the treatmentor prevention of an HPV related disease, wherein the medicament is forintradermal administration.

The sequence of the peptide used in the present invention is notcritical as long as it is derived from an HPV-E2, -E6 and/or -E7 proteinfrom HPV 16 or 18. Preferably, the peptide is chosen in one of the mostimmunogenic regions of these proteins. More preferably, the peptide iscapable of inducing and/or enhancing an HPV-E2, -E6, and/or E7 specificT cell response, and therefore the peptide comprises a specific T cellepitope.

Peptides with a length that exceeds the length of HLA class I and classepitopes (e.g. having a length as indicated herein) are particularlyadvantageous for useas a medicament because they are large enough torequire the phagocytic machinery for antigen uptake as is present inprofessional antigen presenting cells (APC), in particular DC, asexplained in WO 02/070006 and processed in the DC before cell surfacepresentation of the contained HLA class I and class II epitopes takesplace. Therefore, the disadvantageous induction of T cell tolerance (asshown in Toes et al., 1996, PNAS 93:7855 and Toes et al., 1996, J.Immunol. 156:3911), is prevented by the use of peptides having a lengthas indicated herein (as shown in Zwaveling et al., 2002, J. Immunol.169:350). Therefore, in a preferred embodiment a use of the invention isprovided wherein said peptide comprises a sequence capable of activatingan APC. By a sequence capable of activating an APC is meant a sequencewhich is capable of at least partly activating an APC, preferably aprofessional APC. Said activation preferably leads to presentation of atleast one epitope of said peptide at the surface of said APC. In aparticularly preferred embodiment said peptide comprises at least two Tcell epitopes for said antigen. The presence of two T cell epitopes forsaid antigen allows an even more efficient induction and/or enhancementof said antigen specific T cell response.

Preferably, at least one of said epitopes comprises a T-helper cellepitope for said antigen or a cytotoxic T lymphocyte (CTL) epitope forsaid antigen. Having at least one or the other epitope present on thepeptide is favorable. Efficient induction and/or enhancement is achievedwhen said peptide comprises a T-helper activating sequence. By aT-helper activating sequence is meant herein a sequence capable of atleast partly activating a T-helper cell. Said activation preferablyleads to improved induction and/or enhancement of said antigen specificT cell response. In one embodiment said peptide comprises at least oneT-helper cell epitope for said antigen and at least one cytotoxic Tlymphocyte (CTL) epitope for said antigen.

Accordingly, a peptide is preferably used, wherein at least one HLAclass II (T helper cell) epitope and/or at least one HLA class I(cytotoxic T cell) epitope are present within a contiguous amino acidsequence from the amino acid of the HPV E2 or E6 and/or E7 protein froma high risk HPV serotype such as serotype 16, 18, 31, 33, or 45. Morepreferably, the contiguous amino acid sequence present in a peptide usedare from the amino acid of the HPV E2, E6 or E7 protein from HPVserotype 16, 18, 31, or 33, even more preferably from HPV serotype 16 or18 and most preferably from HPV serotype HPV 16. The amino acidsequences of HPV16 and HPV18 E2, E6 and E7 are depicted in SEQ ID No. I,2, 3, 4, 5 and 6 respectively.

Preferably the length of the contiguous amino acid sequence is no morethan 45 amino acids and comprises at least 19 amino acids selected fromthe amino acid sequence of the HPV16 or HPV18 E2, E6 and/or E7 protein(e.g. SEQ ID No. 1, 2, 3, 4, 5, 6), wherein the peptide comprises atleast one HLA class II epitope and/or at least one HLA class I epitope,both from the amino acid sequence of the HPV E2, E6 and/or E7 protein.More preferably, in the peptide at least one HLA class II epitope and/orat least one HLA class I epitope are present within a contiguous aminosequence from the amino acid sequence of the HPV E2, E6 and/or E7protein.

For sake of clarity the peptide used in the invention preferablycomprises at least one HLA class I epitope and/or at least one HLA classII epitope, each of these epitopes are presentable and will bind to thecorresponding specific HLA molecule present on the cells after havingbeen processed as described herein. Each HLA epitope may therefore alsobe named a HLA binding and/or presentable epitope. More preferably, thepeptide used is capable of inducing and/or enhancing an HPV-E2, -E6,and/or E7 specific T cell response, wherein the peptide comprises a Tcell epitope specific for said HPV-E2, -E6, and/or E7 protein. Even morepreferably, the peptide comprises 22-45 contiguous amino acid residuesfrom the HPV E2, or E6 and/or E7 protein.

The length of the contiguous amino acid sequence from the HPV E2, E6and/or E7 protein comprised within the peptide, preferably is comprisedbetween 19-45, 22-45, 22-40, 22-35, 24-43, 26-41, 28-39, 30-40, 30-37,30-35, 32-35 33-35, 31-34 amino acids. In another preferred embodiment,the peptides comprises 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 or more than 45contiguous amino acid residues of the HPV E2, E6 and/or E7 protein. Inanother preferred embodiment, the peptide of the invention consists ofany of the contiguous amino acid sequences from the HPV E2, E6 and/or E7protein as defined herein. The peptides used in the invention may beeasily synthesized and are large enough to be taken up by professionalAPCs, processed by the proteasome and have sufficient physical capacityand length to contain at least one HLA class I and/or one HLA class IIepitope. Accordingly as defined above, m a preferred embodiment, thepeptide comprises a sequence capable of activating an APC.

Alternatively or in combination with former preferred embodiment, thepeptide comprises a T helper activating sequence.

In an even more preferred embodiment, the peptide comprises at least twoT cell epitopes for said antigen. Most preferably, in this peptide atleast one of said epitopes comprises a T-helper cell epitope for saidantigen and/or a cytotoxic T lymphocyte (CTL) epitope for said antigen.

In another preferred embodiment, the antigen present in the peptidederives from the HPV E2, E6 and/or E7 protein or an immunogenic part,derivative and/or analogue thereof. An immunogenic part, derivativeand/or analogue of a protein comprises the same immunogenic capacity inkind not necessarily in amount as said protein itself. A derivative ofsuch a protein can be obtained by preferably conservative amino acidsubstitution.

More preferably, said peptide comprises E2, E6 and/or E7 regions thatwere identified herein as most immunogenic ones. Furthermore, a numberof naturally processed Th-epitopes mapping in this region has alreadybeen identified. Methods included respectively short and long-term PBMCcultures derived from healthy blood donors may be used to identifysuitable peptides. The PBMC cultures may be stimulated with the peptidesto be tested. In parallel, the in vivo induced E2, E6 and/or E7-specificimmunity, as detected by IFNy ELISPOT assays, may be analyzed in healthysubjects as well as subjects diagnosed with HPV I6⁺ lesions.

In a preferred embodiment, the medicament used herein comprises at leasttwo different peptides derived from the HPV-E2, E6 and/or E7 proteins.More preferably, at least three, at least four, at least five, at leastsix, at least seven, at least eight or more peptides are used incombination as a mix or a pool in the medicament. This is advantageoussince vaccination and subsequent protection could be obtained forseveral immunogenic epitopes present within one HPV protein E2, E6 or E7using one single type of vaccine.

Alternatively or in combination with former preferred embodiment, themedicament comprises peptides whose contiguous amino acid sequences arederived from at least two of the HPV E2, E6 and E7 proteins, morepreferably from all three of the HPV E2, E6 and E7 proteins. This isalso advantageous since vaccination and subsequent protection could beobtained for several immunogenic epitopes present within several HPVproteins E2, E6 or E7 using one single type of vaccine.

When the medicament used comprises more than one peptide, thecombination of several peptides may also be named pool or mix ofpeptides.

In a preferred embodiment, the medicament comprises at least one of thefollowing peptides, each peptide comprises or consists of or overlapswith the following sequences derived from E2, E6 or E7 of HPV16: E231-60 or SEQ ID NO:7, E2 46-75 or SEQ ID NO:8, E2 301-330 or SEQ IDNO:9, E2 316-345 or SEQ ID NO:10, E6 1-32 or SEQ ID NO:11, E6 19-50 orSEQ ID NO:12, E6 41-65 or SEQ ID NO:13, E6 55-80 or SEQ ID NO:14, E671-95 or SEQ ID NO:15, E6 85-109 or SEQ ID NO:16, E6 91-122 or SEQ IDNO:17, E6 109-140 or SEQ ID NO:18, E6 127-158 or SEQ ID NO:19, E7 1-35or SEQ ID NO:20, E7 22-56 or SEQ ID NO:21, E7 43-77 or SEQ ID NO:22, E764-98 or SEQ ID NO:23, E2 1-30, E2 16-45, E2 61-90, E2 51-70, E2 61-76,E2 76-105, E2 91-120, E2 106-135, E2 121-150, E2 136-165, E2 151-180, E2166-195, E2 181-210, E2 196-225 E2 211-240, E2 226-255, E2 241-270, E2256-285, E2 271-300, E2 286-315, E2 316-330, E2 311-325, E2 331-365, E2346-355, E2 351-365, E6 1-22, E6 11-32, E6 21-42, E6 31-52, E6 41-62, E651-72, E6 61-82, E6 71-92, E6 81-102, E6 91-112, E6 101-122, E6 111-132,E6 121-142, E6 127-140, E6 131-152, E6 137-158, E7 1-22, E7 11-32, E721-42, E7 30-50, E7 31-52, E7 35-50, E7 41-62, E7 50-62, E7 51-72, E735-77, E7 61-82, E7 71-92, E7 77-98, of which E2 31-60 or SEQ ID NO:7,E2 46-75 or SEQ ID NO:8, E2 301-330 or SEQ ID NO:9, E2 316-345 or SEQ IDNO:10, E6 1-32 or SEQ ID NO:11, E6 19-50 or SEQ ID NO:12, E6 41-65 orSEQ ID NO:13, E6 55-80 or SEQ ID NO:14, E6 71-95 or SEQ ID NO:15, E685-109 or SEQ ID NO:16, E6 91-122 or SEQ ID NO:17, E6 109-140 or SEQ IDNO:18, E6 127-158 or SEQ ID NO:19, E7 1-35 or SEQ ID NO:20, E7 22-56 orSEQ ID NO:21, E7 43-77 or SEQ ID NO:22, E7 64-98 or SEQ ID NO:23 aremost preferred. The sequence of each of these peptides can be deducedfrom the full length sequence of the corresponding E2, E6 or E7 of HPV16as depicted in SEQ ID NO: 1, 2, or 3.

In the context of the invention, overlapping means that the sequence ofthe peptide used partially or totally overlaps with the given sequence.Preferably, overlapping means partially overlapping. Partiallypreferably means that the overlap is of one or more amino acids at the5′end and/or at the 3′end of the peptide sequence, more preferably oftwo or more amino acids at the 5′end and/or at the 3′end, or more. It isalso preferred that the overlap is of one or more amino acids at the5′end and/or two or more amino acids at the 3′end of the peptidesequence or vice versa. The skilled person will understand that allkinds of overlaps are encompassed by the present invention as long asthe obtained peptide exhibits a desired immunogenic activity as earlierdefined herein.

In another preferred embodiment, the peptides used in the medicament arederived from the sequences given above by conservative amino acidsubstitution. More preferably, the medicament comprises at least two ofthe peptides as specifically mentioned above are used, or at least threeor at least four, or at least five, or at least six or more.

In most preferred embodiments, the medicament comprises at least one ofthe following pools of peptides, wherein each peptide comprises orconsists of or overlaps with the following sequences:

pool 1: E2 31-60 and/or E2 46-75, and/orpool 2: E2 301-330 and/or E2 316-345, and/orpool 3: E2 31-60 and/or E2 46-75, and/or E2 301-330 and/or E2 316-345,and/orpool 4: E6 1-32 and/or E6 19-50, and/orpool 5: E6 41-65, E6 55-80 and/or E6 71-95, and/orpool 6: E6 85-109, and/or E6 91-122 and/orpool 7: E6 109-140 and/or E6 127-158, and/orpool 8: E6 1-32 and/or E6 19-50, and/or E6 41-65, and/or E6 55-80 and/orE6 71-95, and/or E6 85-109, and/or E6 91-122 and/or E6 109-140 and/or E6127-158, and/orpool 9: E7 1-35 and/or E7 22-56, and/orpool 10: E7 43-77, and/or E7 64-98, and/orpool 11: E7 1-35 and/or E7 22-56, and/or E7 43-77, and/or E7 64-98,and/orpool 12: pool 3 and pool 8 as defined above, and/orpool 13: pool 8 and pool 11 as defined above, and/or pool 14: pool 3 andpool 11 as defined above, and/or pool 15: pool 3, pool 8 and pool 11 asdefined above. Preferably, the class II CD4+Th cell epitope comprised ina peptide present in the medicament is capable of activating a CD4+Thcell in the patient with HPV-induced disease and/or a healthy subject.The activation is preferably assessed ex vivo or in vivo, morepreferably in the patient with HPV-induced disease of whom theHPV-infected/transformed cells express the given antigen. Mostpreferably, the HLA class II epitope is capable of activating a CD4+Thmemory response, i.e. activation of a CD45 RO-positive CD4+ T-helpercell. This will lead, by virtue of the ‘licence to kill’ signal throughCD40-triggering of DC (Lanzavecchia, 1998, Nature 393:413), to a morerobust CD8⁺ effector and memory T-cell response. The art currently knowsmany ways of generating a peptide. The invention is not limited to anyform of generated peptide as long as the generated peptide comprises aminimal T cell epitope. By way of example, a peptide present in themedicament can be obtained from protein E2, E6 or E7, synthesized invitro or by a cell, for instance through an encoding nucleic acid. Apeptide used in the medicament can be present as a single peptide orincorporated into a fusion protein. In one embodiment said peptide isflanked by processing sites allowing processing of said peptide inside acell such as to allow transport and/or incorporation into an MHCmolecule on the surface of said cell. In a preferred embodiment apeptide used in the medicament is after processing capable of complexingwith an MHC class II molecule. MHC class II restricted T-cell immunityis currently considered to be important in eradication of for instancetumor cells although said tumor cells often do not express MHC class IImolecules. Peptides used in the medicament are particularly well suitedfor eliciting, inducing and/or stimulating both MHC class I and MHCclass II dependent T cells.

A peptide used in the medicament may further be modified by deletion orsubstitution of one or more amino acids, by extension at the N- and/orC-terminus with additional amino acids or functional groups, which mayimprove bio-availability, targeting and uptake by professional APC, orcomprise or release immune modulating substances that provide adjuvantor (co)stimulatory functions. The optional additional amino acids at theN- and/or C-terminus are preferably not present in the correspondingpositions in the HPV E2, E6 and/or E7 amino acid sequence, morepreferably they are not from the E2, E6, or E7 amino acid sequence (SEQID NO. 1, 2, 3, 4, 5, 6).

In a further preferred embodiment, the medicament does not comprise anadjuvant. More preferably, the medicament does not comprise an adjuvantcurrently known to be associated with at least one of the followingundesired side effects such as local swelling at the site of injection,red swollen hands, fever, vomiting, joint pain, a general illnessfeeling similar to symptoms experienced during an infection withinfluenza. Even more preferably, the adjuvant is not of the type of anoil-in water emulsions such as incomplete Freund's adjuvant or IFA,Montanide ISA-51 or Montanide ISA 720 (Seppic France). Even morepreferably, the adjuvant does not have a depot function and/or isbiological degradable. A depot function preferably means that thepeptide is contained for a long time in the injection site and onlyleaks out over a long time period. Preferably, a long time period is ofat least one month, more preferably at least two, or three months. Evenmore preferably, the adjuvant is not Montanide ISA-51 (Seppic France).

In another further preferred embodiment, the medicament consists of oneor more peptides as earlier defined herein and an inert pharmaceuticallyacceptable carrier and/or excipients. The inert pharmaceuticallyacceptable carrier and/or excipients preferably is inert in the sensethat it does not invoke an immune response and/or an inflammatoryresponse or any of the undesired side effects described above foradjuvants. Formulation of medicaments, and the use of pharmaceuticallyacceptable excipients are known and customary in the art and forinstance described in Remington; The Science and Practice of Pharmacy,21^(nd) Edition 2005, University of Sciences in Philadelphia. Themedicament used in the invention is formulated to be suitable forintradermal administration or application. Intradermal is known to theskilled person. In the context of the invention, intradermal issynonymous with intracutaneous and is distinct from subcutaneous. A mostsuperficial application of a substance is epicutaenous (on the skin),then would come an intradermal application (in or into the skin), then asubcutaneous application (in the tissues just under the skin), then anintramuscular application (into the body of the muscle). An intradermalapplication is usually given by injection. An intradermal injection of asubstance is usually done to test a possible reaction, allergy and/orcellular immunity to it. A subcutaneous application is usually alsogiven by injection: a needle is injected in the tissues under the skin.

In another further preferred embodiment, since the medicament used inthe invention does not comprise any adjuvant such as Montanide ISA-51,it means the formulation of the medicament is more simple: an oil-waterbased emulsion is preferably not present in the medicament used.Accordingly, the medicament used in the invention does not comprise anadjuvant such as Montanide ISA-51 and/or does not comprise anoil-in-water based emulsion. Therefore, in a preferred embodiment, themedicament used in the invention is a buffered aqueous solutions atphysiological ionic strength and/or osmolarity, such as e.g. PBS(Phosphate Buffer Saline) comprising or consisting of one or morepeptide as defined earlier herein. The skilled person knows how toprepare such a solution.

The medicament as used in the invention has another advantage, which isthat by intradermally administering low amounts of a peptide as earlierherein defined, an immunogenic effect may still be achieved. The amountof each peptide used is preferably ranged between 1 and 1000 μg, morepreferably between 5 and 500 μg, even more preferably between 10 and 100μg.

The skilled person would know how to test whether the concentration ofpeptide envisaged is immunogenic. Preferably, PBMC are in vitrostimulated with different concentrations of a peptide to be tested asillustrated in WO 02/070006. In example, an immunogenic effect isreached when peptide-stimulated PBMC start to proliferate at least 2times stronger, and/or produce at least 2-fold more cytokine, and/orupregulate activation markers (e.g. CD25, HLA-DR, CD69, CD154, CD137)than non-stimulated PBMC. Alternatively, a skin test is performed as inthe examples. Briefly, the chosen peptide is intracutaneously injected,preferably 0.05 ml of about 0.1 to about 0.4 mg/ml, more preferably 0.2mg/ml peptides in about 10-20%, more preferably about 16% DMSO (v/v) in20 mM isotonic phosphate buffer (10 μg/peptide). The peptides areinjected separately at individual skin test sites of the upper arm.

In another preferred embodiment, the medicament comprises a peptide asearlier defined herein and at least one adjuvant, said adjuvant beingnot formulated in an oil-in water based emulsion and/or not being of anoil-in-water emulsion type as earlier defined herein. This type ofmedicament may be administered as a single administration.Alternatively, the administration of a peptide as earlier herein definedand/or an adjuvant may be repeated if needed and/or distinct peptidesand/or distinct adjuvants may be sequentially administered. It isfurther encompassed by the present invention that a peptide of theinvention is administered intradermally whereas an adjuvant as definedherein is sequentially administered. The adjuvant may be intradermallyadministered. However any other way of administration may be used forthe adjuvant.

Particularly preferred adjuvants are those that are known to act via theToll-like receptors and/or via a RIG-I (Retinoic acid-inducible gene I)protein and/or via an endothelin receptor. Adjuvants that are capable ofactivation of the innate immune system, can be activated particularlywell via Toll like receptors (TLR's), including TLR's 1-10. Compoundscapable of activating TLR receptors and modifications and derivativesthereof are well documented in the art. TLR1 may be activated bybacterial lipoproteins and acetylated forms thereof, TLR2 may inaddition be activated by Gram positive bacterial glycolipids, LPS, LPA,LTA, fimbriae, outer membrane proteins, heatshock proteins from bacteriaor from the host, and Mycobacterial lipoarabinomannans TLR3 may beactivated by dsRNA, in particular of viral origin, or by the chemicalcompound poly(I:C). TLR4 may be activated by Gram negative LPS, LTA,Heat shock proteins from the host or from bacterial origin, viral coator envelope proteins, taxol or derivatives thereof, hyaluronancontaining oligosaccharides and fibronectins. TLR5 may be activated withbacterial flagellae or flagellin. TLR6 may be activated by mycobacteriallipoproteins and group B Streptococcus heat labile soluble factor(GBS-F) or Staphylococcus modulins. TLR7 may be activated byimidazoquinolines. TLR9 may be activated by unmethylated CpG DNA orchromatin IgG complexes. In particular TLR3, TLR7 and TLR9 play animportant role in mediating an innate immune response against viralinfections, and compounds capable of activating these receptors areparticularly preferred for use in the invention. Particularly preferredadjuvants comprise, but are not limited to, synthetically producedcompounds comprising dsRNA, poly(I:C), unmethylated CpG DNA whichtrigger TLR3 and TLR9 receptors, IC31, a TLR9 agonist, IMSAVAC, a TLR 4agonist. RIG-1 is known to be activated by ds-RNA just like TLR3(Immunity (2005), 1:19-28). In another preferred embodiment, theadjuvants are physically linked to a peptide as earlied defined herein.Physical linkage of adjuvants and costimulatory compounds or functionalgroups, to the HLA class I and HLA class II epitope comprising peptidesprovides an enhanced immune response by simultaneous stimulation ofantigen presenting cells, in particular dendritic cells, thatinternalize, metabolize and display antigen. Another preferred immunemodifying compound is a T cell adhesion inhibitor, more preferably aninhibitor of an endothelin receptor such as BQ-788 (Buckanovich R J etal, Ishikawa K, PNAS (1994) 91:4892). BQ-788 isN-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine.However any derivative of BQ-788 or modified BQ-788 compound is alsoencompassed within the scope of this invention.

Furthermore, the use of APC (co)stimulatory molecules, as set out inWO99/61065 and in WO03/084999, in combination with a peptide present inthe medicament used in the invention is preferred. In particular the useof 4-1-BB and/or CD40 ligands, agonistic antibodies, OX40 ligands orfunctional fragments and derivates thereof, as well as syntheticcompounds with similar agonistic activity are preferably administeredseparately or combined with a peptide present in the medicament tosubjects to be treated in order to further stimulate the mounting anoptimal immune response in the subject.

In a preferred embodiment, the adjuvant comprises a TLR (3,4,7,8,9)ligand such as monophosphoryl lipid A and/or CpG nucleic acid, and/or anAPC-costimulatory molecule such as a CD40 ligand, agonistic antibodiesor functional fragments and derivates thereof, and/or GM-CSF.

In another preferred embodiment, to promote the presentation of apeptide by a professional antigen presenting cell or dendritic cells,the medicament comprising a peptide further comprises a DC-activatingagent as a TLR ligand as earlier mentioned herein.

In a preferred embodiment, the medicament, which is a vaccine isadministered to a human or animal. In a more preferred embodiment, thehuman or animal is suffering from or at risk of suffering from a HPV(persistent) related disease. Said HPV related disease is selected froman HPV infection, an HPV associated malignancy, a CervicalIntra-epithelial Neoplasia (CIN), a Vulva Intra-epithelial Neoplasia(VIN), an Anal Intra-epithelial Neoplasia (AIN), a VaginalIntra-epithelial Neoplasia (VAIN), Penile Intra-epithelial Neoplasia(PIN), cervical cancer, head and neck cancer, in particularoropharyngeal cancer and tonsillar cancers, penile cancer, anal cancer,vaginal cancer and vulvar cancer.

Preferably, said HPV related disease is at least in part treatable orpreventable by inducing and/or enhancing said immune response.

The method of the invention is therefore very suited for providing asubject with immunity against said antigen and/or for enhancing saidimmunity. Methods of the invention are suitable for any purpose thatother immunization strategies are used for. Old immunizations are usedfor vaccination purposes, i. e. for the prevention of disease. However,methods of the invention are not only suitable for preventing disease.Methods can also be used to treat existing disease, of course with thelimitations that the disease is treatable by inducing and/or enhancingantigen specific T cell immunity. This feature can be used to treat, forinstance, diseases associated with viral infections such as HPVinfection, such as some cancers. In a preferred embodiment said human oranimal is suffering from or at risk of suffering from a disease such asan HPV infection that is at least in part treatable or preventable byinducing and/or enhancing said immune response. Preferably said diseasecomprises a HPV viral disease and/or cancer.

The intradermal administration of a peptide is very attractive since theinjection of the vaccine is realized at or as close by as possible tothe site of the disease resulting in the local activation of the diseasedraining lymph node, resulting in a stronger local activation of theimmune system. In particular for VIN, VAIN, AIN, PIN, Penile cancer,Vulva cancer, Anal cancer, Head and Neck cancers.

In a preferred embodiment, the intradermal administration is carried outdirectly at the site of the lesion or disease. At the site of the lesionis herein understood to be within less than 5, 2, 1, 0.5, 0.2 or 0.1 cmfrom the site of the lesion.

Upon intradermally administering a medicament as defined herein, notonly Th2 but also Th1 responses are triggered. This is surprising sinceit was already found that cutaneous antigen priming via gene gun lead toa selective Th2 immune response (Alvarez D, et al, 2005). Furthermore,the immune response observed is not only restricted to the skin as couldbe expected based on Alvarez D., et al. We demonstrate that specific Tcells secreting IFN^(Y) circulate through the secondary lymph system asthey are detected in the post challenged peripheral blood.

Another crucial advantage of the medicament of the invention is thatrelatively low amounts of peptides may be used, in one single shot, in asimple formulation and without any adjuvant known to give undesiredside-effects as Montanide ISA-51. Without wishing to be bound by anytheory, we believe the HPV intradermal peptide(s) used in the inventionspecifically and directly targets the epidermal Langerhans cells (LC)present in the epithelium. Langerhans cells are a specific subtype of DCwhich exhibit outstanding capacity to initiate primary immune responses(Romani N., et al,). These LC may be seen as natural adjuvants recruitedby the medicament used in the invention.

In another preferred embodiment, the invention relates to the use of apeptide derived from HPV-E2, -E6 and/or -E7 protein for the manufactureof a medicament for the treatment or prevention of an HPV relateddisease, wherein the medicament is for intradermal administration asearlier defined and wherein in addition a peptide derived from HPV-E2,-E6 and/or -E7 protein is further used for the manufacture of amedicament for the treatment or prevention of an HPV related disease,wherein the medicament is for subcutaneous administration.

The medicament for intradermal administration has already been definedherein. The peptide used for subcutaneous administration is the same asthe one used for intradermal administration and has already been definedherein. The skilled person knows how to formulate a medicament suitedfor subcutaneous administration. Preferably, the medicament suited forsubcutaneous administration comprises a peptide as already hereindefined in combination with an adjuvant. Preferred adjuvants havealready been mentioned herein. Other preferred adjuvants are of the typeof an oil-in water emulsions such as incomplete Freund's adjuvant orIFA, Montanide ISA-51 or Montanide ISA 720 (Seppic France). In a furtherpreferred embodiment, the medicament suited for subcutaneousadministration comprises one or more peptides, an adjuvant both asearlier defined herein and an inert pharmaceutically acceptable carrierand/or excipients all as earlier defined herein. Formulation ofmedicaments, and the use of pharmaceutically acceptable excipients areknown and customary in the art and for instance described in Remington;The Science and Practice of Pharmacy, 21^(nd) Edition 2005, Universityof Sciences in Philadelphia. The second medicament used in the inventionis formulated to be suitable for subcutaenous administration.

In this preferred embodiment, the medicament suited for intradermaladministration may be simultaneously administered with the medicamentsuited for subcutaneous administration. Alternatively, both medicamentmay be sequentially intradermally and subsequently subcutaneouslyadministered or vice versa (first subcutaneous administration followedby intradermal administration). In this preferred embodiment as inearlier preferred embodiment dedicated to the intradermaladministration, the intradermal and/or subcutaneous administration of apeptide as earlier herein defined and/or of an adjuvant may be repeatedif needed and/or of distinct peptides and/or of distinct adjuvants maybe sequentially intradermally and/or subcutaneously administered. It isfurther encompassed by the present invention that a peptide of theinvention is administered intradermally and/or subcutaneously whereas anadjuvant as defined herein is sequentially administered. The adjuvantmay be intradermally and/or subcutaneously administered. However anyother way of administration may be used for the adjuvant.

We expect the combination of an intradermal and a subcutaneousadministration of a medicament according to the invention isadvantageous. DC in the epidermis are clearly different from DC in thedermis and in the subcutis. The intracutaneous (intradermal)immunization will cause antigen processing and activation of epidermalDC (Langerin-positive langerhans cells) that through their dendriticnetwork are in close contact with the keratinocytes. This will alsooptimally activate inflammatory pathways in the interactions betweenLangerhans cell and keratinocytes, followed by trafficking of antigenloaded and activated Langerhans cell to the skin-draining lymph nodes.

The subcutaneous administration will activate other DC subsets, thatwill also become loaded with antigen and travel independently to theskin-draining lymph nodes. Conceivably, the use of a medicament whichmay be administered both intradermally and subcutaneously may lead to asynergistic stimulation of T-cells in these draining nodes by thedifferent DC subsets.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition, reference to an element by the indefinitearticle “a” or “an” does not exclude the possibility that more than oneof the element is present, unless the context clearly requires thatthere be one and only one of the elements. The indefinite article “a” or“an” thus usually means “at least one”.

The invention is further illustrated by the following examples, whichshould not be construed for limiting the scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

An overview of the number, day of appearance and injected antigen thatinduced a positive skin reactions in the group of 19 healthy donors (HD)and 17 patients (P) with history of cervical neoplasia. Skin reactionswere considered positive when papules greater then 2 mm in diameterarose no less then 2 days after injection. The indicated layout is usedfor the 8 peptide pools, the first and last amino acid in the protein ofthe peptide pool used is indicated. The layout printed in bold indicatesat least one positive reaction within this timeframe; a filled squarerepresents a new developed, positive skin reaction to the indicatedpeptide pool.

FIG. 2

Detection of HPV16 specific T cells by IFNy ELispot in the pre-challengeblood sample of healthy donors is significantly correlated with theappearance of an early (<13 days) positive skin reaction to therecognized peptide pool (p=0.0003, two tailed Fisher's Extract test).Specific responses were calculated by subtracting the mean number ofspots+2xSD of the medium control from the mean number of spots inexperimental wells. The number of specific spots per 100.000 PBMC isgiven. Responses were considered positive if peptide pool specific Tcell frequencies were 2′: 5 in 100.000 PBMCs.

FIGS. 3a and 3b

3a. Association between the appearance of a positive skin reaction andthe simultaneous detection (IFN^(Y) ELispot) of circulating HPV16specific T cells in the post-challenge blood sample of healthy donors(p<0.0001, two tailed Fisher's exact test). From a total of 88 skintests, 39 were positive. Twenty-five of these 39 reactions wereassociated with a positive reaction in ELispot (T cell frequency 2′: 5in 100.000 PBMCs). Of the 49 skin test sites that did not show a skinreaction, 10 were associated with a positive ELlspot.

3b. Example of a healthy donor (HD 10) displaying a positive skinreaction at day 14 to peptide pool 6 (E6109-140, E6127-1ss) (leftpanel). Punch biopsy of the positive skin reaction site (right panel).

FIG. 4

A. HPV16 specific T cell responses detected by IFNy ELispot in thepost-challenge blood sample of healthy donors displaying a positive skinreaction. The mean number of spots per 100.000 PBMCs are depicted.Memory response mix (MRM) was used as a positive control. The filled barindicates the positive skin reaction site of which a punch biopsy wastaken and put in to culture.

B. T lymphocytes exfiltrating from punch biopsies were, after a 14- to28 day period of cytokine driven expansion, tested for their capacity toproliferate upon stimulation with monocytes pulsed with peptides (10μg/ml)-as injected in the skin test- or with protein (20 μg/ml).Phytohemagglutinine (PHA) served as a positive control. Proliferationwas measured by [3H]thymidine incorporation and a proliferative responsewas defined specific as the stimulation index (SI) 2′: 3. Healthy donor17 (HD17) is an example of a positive skin reaction site consisting ofnon specific T cells.

C. Supematants of the proliferative responses in B were analysed for thepresence of IFNy, interleukin 4 (IL4), IL5 and tumor necrosis factor a,IL2, IL10 (not shown) by cytometric bead array. Cutoff values were basedon the standard curves of the different cytokines (100 pg/ml IFNy and 20pg/ml for the remaining cytokines). Antigen-specific cytokine productionwas defined as a cytokine concentration above cutoff level and >2× theconcentration of the medium control. Healthy donor 15 (HD15) displays ahigh background level ofIL5, but is increased >2× after antigenstimulation.

FIG. 5.

T cell culture of the skin biopsy of pool 4 (E641-6s, E6ss-so, E611-9s)of healthy donor 15 (HD 15) consists of both HPV 16 specific CD4+ andCDS+ T cells. The specificity of the culture was tested in anintracellular cytokine staining (ICS) against the protein (20

μg/ml) and the peptides (10 μg/ml) corresponding with the injected skintest. Remarkably, in 3 out of 4 biopsies CDS+ HPV16-specific T cellswere detected.

EXAMPLES Materials and Methods Study Design

A cross-sectional pilot study to analyse HPV16 E2-, E6-, and E7-specificT-cell responses as measured by intradermal injection of pools ofclinical grade HPV16 peptides in the upper arm was performed in patientswith HPV-related disorders of the cervix and in healthy individuals.Since a delayed type hypersensitivity reaction represents a memoryT-cell response, there was no prerequisite for HPV16-positivity at thetime of analysis.

Subjects

Seventeen women (P) with history of cervical carcinoma (n=12) or CIN(n=5) and a group of nineteen healthy individuals (HD) participated inthis study after providing informed consent. The clinicalcharacteristics of the patients, including HPV status, are summarized inTable 1. The age of the patients ranged from 28-72 years (median age, 46years). The group of healthy individuals displayed a median age of 31years old (range, 20-51 years) and was comprised of 80% women and 20%males. Peripheral blood mononuclear cells (PBMCs) were obtained from allsubjects immediately before administration of the skin test. The lateappearance of positive skin tests in healthy individuals resulted in theisolation of a second blood sample from 11 of 19 healthy volunteers. Thestudy design was approved by the Medical Ethical Committee of the LeidenUniversity Medical Centre.

DTH Skin Test

Skin tests, based on Delayed Type Hypersensitivity reactions (DTH), canbe used as a sensitive and simple method for in vivo measurement ofHPV-specific cellular immune responses (Hopfl, 2000; Hopfl, 1991). Theskin test preparations consisted of 8 pools of long clinical-gradesynthetic peptides spanning the whole HPV 16 E6 and E7 protein and themost immunogenic regions of HPV 16 E2 protein (de Jong, 2004). Theseclinical grade peptides were produced in the interdivisionalGMP-Facility of the LUMC. Each pool of the skin test consisted of 2 or 3synthetic peptides, indicated by the first and last amino acid of theregion in the protein covered by the peptides. Pool 1: E231-60, E246-1s,Pool 2: E2301-330, E2316-34s, Pool 3: E61-31, E619-so, Pool 4: E641-6s,E6ss-8o, E611-9s, Pool 5: E68s-109, E691-122, Pool 6: E6109-140,E6121-1s8, Pool 7: E71-3s, E722-s6, Pool 8: E743_77, E764-98. Perpeptide pool 0.05 ml of 0.2 mg/ml peptides in 16% DMSO in 20 mM isotonicphosphate buffer (10 μg/peptide) was injected intracutaneously. Thepools of peptides and a negative control (dissolvent only) were injectedseparately at individual skin test sites of the upper arm. Skin testsites were inspected at least three times, at 72 hours and 7 days afterinjection (Hopfl) of the peptides and at 3 weeks following the firstreport of a very late skin reaction in one of the first healthysubjects. Reactions were considered positive when papules greater than 2mm in diameter arose no less than 2 days after injection. From positiveskin reaction sites punch biopsies (4 mm) were obtained, cut in smallpieces and cultured in IMDM containing 10% human AB serum, 10% TCGF and5 ng/ml IL7 and IL 15 to allow the emigration of lymphocytes out of theskin tissue. After 2 to 4 weeks of culture the expanded T cells wereharvested and tested for their HPV-specific reactivity.

Antigen for In Vitro Immune Assays

A set of peptides, similar to the peptides used in the skin test, wereused for T-cell stimulation assays and IFNy-ELISPOT assays. The four HPV16 E2 peptides consisted of 30-mer peptides overlapping 15 residues, HPV16 E6 consisted of 32-mers and HPV 16 E7 of 35-mers, both overlapping 14residues. The peptides were synthesized and dissolved as previouslydescribed (van der Burg, 1999). Notably, in the IFN: ELISPOT assayspeptide pool 4 and 5 slightly differed from the peptide pools used inthe skin test, pool 4 contained peptides E6₃₇₋₆₈, E6₅₅₋₈₆, E6₇₃₋₁₀₄ andpool 5 comprised peptides E6₇₃₋₁₀₄, E6₉₁₋₁₂2.Memory response mix (MRM 50×), consisting of a mixture of tetanus toxoid(0.75 Limus flocculentius/ml; National Institute of Public Health andEnvironment, Bilthoven, The Netherlands), Mycobacterium tuberculosissonicate (5 μg/ml; generously donated by Dr. P. Klatser, Royal TropicalInstitute, Amsterdam, The Netherlands), and Candida albicans (0.15mg/ml, HAL Allergenen Lab., Haarlem, The Netherlands) was used as apositive control. Recombinant HPV 16 E2, E6 and E7 proteins wereproduced in recombinant Escherichia coli as described previously (vander Burg, 2001).

Antigen-Specific Th Cells by IFNγ ELISPOT

The presence of HPV 16-specific Th Cells was analyzed by ELISPOT asdescribed previously (van der Burg, 2001) Briefly, fresh PBMCs wereseeded at a density of 2×10⁶ cells/well of a 24-well plate (Costar,Cambridge, Mass.) in 1 ml of IMDM (Bio Whittaker, Verviers, Belgium)enriched with 10% human AB serum, in the presence or absence of theindicated HPV 16 E2, E6 and E7 peptide pools. Peptides were used at aconcentration of 5 μg/ml/peptide. After 4 days of incubation at 37° C.,PBMCs were harvested, washed, and seeded in four replicate wells at adensity of 10⁵ cells per well in 100 μl IMDM enriched with 10% FCS in aMultiscreen 96-well plate (Millipore, Etten-Leur, The Netherlands)coated with an IFNy catching antibody (Mabtech AB, Nacha, Sweden).Further antibody incubations and development of the ELISPOT wasperformed according to the manufacturer's instructions (Mabtech). Spotswere counted with a fully automated computer-assisted-video-imaginganalysis system (Bio Sys). Specific spots were calculated by subtractingthe mean number of spots+2xSD of the medium control from the mean numberof spots in experimental wells (van der Burg, 2001).

T Cell Proliferation Assay

T-cell cultures of the skin biopsies were tested for recognition of thespecific peptides and protein in a 3-day proliferation assay (van derBurg, 2001). Briefly, autologous monocytes were isolated from PBMCs byadherence to a flat-bottom 96-well plate during 2 h in X-vivo 15 medium(Cambrex) at 37° C. The monocytes were used as APCs, loaded overnightwith 10 μg/ml peptide and 20 μg/ml protein. Skintest—infiltrating-lymfocytes were seeded at a density of 2-5×10⁴cells/well in IMDM supplemented with 10% AB serum. Medium alone wastaken along as a negative control, phytohemagglutinine (0.5 μg/ml)served as a positive control. Proliferation was measured by[3H]thymidine (5 μCi/mmol) incorporation. A proliferative response wasdefined specific as the stimulation index (SI) ≧3. Supernatants of theproliferation assays were harvested 48 hours after incubation for theanalysis of antigen-specific cytokine production.\Analysis of Cytokines Associated with HPV16-Specific ProliferativeResponsesThe simultaneous detection of six different Th 1 and Th2 cytokines:IFNy, tumor necrosis factor a, interleukin 2 (IL2), IL4, IL5 and IL 10was performed using the cytometric bead array (Becton Dickinson)according to the manufacturer's instructions. Cut-off values were basedon the standard curves of the different cytokines (100 pg/ml IFNγ and 20pg/ml for the remaining cytokines). Antigen-specific cytokine productionwas defined as a cytokine concentration above cutoff level and >2× theconcentration of the medium control (de Jong, 2004).

Intracellular Cytokine Staining (ICS)

The specificity and character of the T cell cultures derived frompositive skin reaction sites was tested by ICS as reported previously(de Jong, 2005). Briefly, skin test infiltrating lymphocytes wereharvested, washed and suspended in IMDM+10% AB serum and 2-5×10⁴ cellswere added to autologous monocytes that were pulsed overnight with 50 μlpeptide (10 μg/ml) or protein (20 μg/ml) in X vivo medium. Medium alonewas taken along as a negative control, phytohemagglutinine (0.5 μg/ml)served as a positive control. Samples were simultaneously stained withFITC-labelled mouse-antihuman IFNγ (0.5 g/ml, BD PharMingen),PE-labelled mouse-antihuman IL5 (0.2 mg/ml, BD PharMingen), APC-labelledanti-CD4 (BD Bioscience) and PerCP-labelled anti-CDS (BD Bioscience).After incubation at 4° C., the cells were washed, fixed with 1%paraformaldehyde and analyzed by flow cytrometry (FACS scan, BDBiosciences)

Statistical Analysis

Fisher's Exact test (2-tailed) was used to analyze the relationshipbetween the detection of IFNy-producing HPV-specific T-cells in PBMC,the presence of a skin test reaction or the presence of HPV-specificT-cells in skin biopsies, as well as differences between patients andhealthy controls with respect to the size or the number of the skinreactions within these groups. Statistical analyzes w e r e performedusing Graphpad Instat Software (version 3.0) and Graphpad Prism 4.

Results

Skin Reactions to Intracutaneous Injection with HPV 16 E2, E6- and E7PeptidesWe studied skin reactions in both healthy subjects and patients with HPVinduced disease after intracutaneous injection with HPV16 E2, -E6 and-E7 peptides. Positive skin reactions appeared as flat reddish papulesof 2 to 20 mm of diameter, arising within 2 to 25 days after injection.A positive skin reaction was detected in 46 of the 152 skin tests in thecontrol group and in 30 out of 136 skin test sites in the patient group.The size of the skin reactions did not differ between the two groups.Over all, each peptide-pool in the skin test could give rise to apositive skin reaction. Most frequently reactions against E2₃₁₋₇₅ (10out of 19 subjects), E6₃₇₋₁₀₄ (9/16) and E7₄₃₋₉₈ (7/19) were observed inthe control group. This reaction pattern resembles that of what wepreviously observed in PBMC (de Jong, 2002; Welters, 2003), as well asthe pattern observed in the patient group (FIG. 1).The time for skin reactions to appear, differed considerably between thegroup of healthy volunteers and patients. A classical DTH reaction,within 24 to 72 hours after injection, was observed in only three cases,2 patients and 1 healthy control (FIG. 1). The majority of skinreactions in the patient group developed within 2 to 20 days, reaching amaximum after one week (FIG. 1). Notably, 5 out of 9 patients withhistory of a HPV16 related disease, showed a positive reaction within 8days. However, within the group of controls most of the skin reactionswere detected between day 13-25.Skin Reactions in Healthy Donors are Associated with Higher Frequenciesof HPV 16-Specific T-Cells in the Peripheral Blood.In order to compare the results of the skin test with the presence ofcirculating HPV16-specific type 1 T cells, an IFNγ ELispot assay wasperfomled with PBMC's collected before the intradermal peptide-challengewas given. In 5 out of 19 healthy volunteers we were able to detect aHPV16-specific immune response by IFNy-ELispot. The detection of 2′: 5circulating HPV16-specific T-cells per 100.000 PBMC in the pre-challengeblood sample of healthy individuals was associated with an early (:S 13days) positive skin reaction to the same peptide sequence (p=0.0003, twotailed Fisher's exact test; FIG. 2). No HPV16-specific circulatingT-cells were detected in the pre-challenge blood sample healthy donorsto peptides that induced a late positive skin reaction (14 to 25 days).This suggests that the frequency of circulating antigen-specific cellsdetermine the delay time for skin reactions to appear. In order toassess the frequency of HPV-specific T-cells at the time that a lateskin reaction appeared additional blood samples from 11 healthyvolunteers were collected. In these individuals 39 out of 88 skin testswere positive. In 25 of the 39 positive skin reactions and in 10 of 49negative skin reactions 2′: 5 HPV 16-specific T-cells were detected per100.000 PBMC. At this point a significant correlation was found betweenthe detection of circulating HPV-specific IFNγ-producing T-cells in thepost-challenged blood sample and the presence of a skin reaction(p<0.0001, Fisher's exact test; FIG. 3a ). This shows that the frequencyof HPV16-specific T cells in the blood of healthy volunteers issignificantly higher following an intradermal challenge with HPV16peptide and indicates that intracutaneous injection of peptide antigensenhances the number of HPV16-specific T cells in the blood of healthyvolunteers.

Biopsies of Positive Skin Reaction Sites Consist of Both Th1/Th2-CD4+and CDS+ HPV16-Specific T Cells.

Approximately 25% of the positive skin reactions of healthy volunteerswere not associated with the detection of HPV16-specific IFNγ-producingT-cells in the blood, suggesting that other, non IFNγ-producing types ofT-cells may infiltrate the skin after intradermal injection of HPV16peptides.In order to characterize the cells in a positive skin reaction sitepunch biopsies were taken, (FIG. 3b ). In total, S biopsies were takenfrom different positive skin reaction sites of 7 healthy controls andcultured with a cocktail of cytokines that allowed the outgrowth ofT-cells in vitro without antigenic stimulans. In 7 of S cases, T-cellsex-filtrated the tissue and expanded within 3-4 weeks. The expandedT-cells were tested for their specificity in a short term proliferationassay. FIG. 4 shows examples of T-cell cultures that specificallyproliferated upon stimulation with autologous monocytes pulsed with thepool of peptides, also injected in this site during the skin test (HD2,HDIO, HD15) as well as to monocytes pulsed with HPV16 E6 protein (FIGS.4, A and B). This indicates that these T-cells were capable ofrecognizing their cognate HLA-peptide complexes after the antigen wasnaturally processed and presented. Analysis of the supematants of theseproliferative T-cell cultures revealed a mixed Th1/Th2 cytokine profilein that the HPV16-specific T-cells produced IFNγ, IL-4 and IL-5 (FIG. 4,C).In each case that HPV-specific T-cells were detected in the biopsyculture (4 out of S) this coincided with the detection of circulatingHPV16-specific IFNγ-producing T-cells in the post-challenge blood sampleby ELispot (compare FIGS. 4, A and B). In 3 of the other 4 positive skinreaction biopsies (HD2, HD 17, HD 1S) the T-cells did not respond toHPV16 peptides (FIG. 4; HD17) and in one case no T-cells ex-filtratedthe tissue at all (HD13). In these 4 cases we were not able to detectcirculating HPV16-specific IFNγ-producing T-cells in the post-challengeblood sample.Co-staining of the biopsy-T cells by CD4 and CDS cell surface markersshowed that not only HPV16-specific CD4+ but also HPV16-specific CDS+ Tcells infiltrated the skin site upon intradermal challenge with HPV16peptide (FIG. 5). Overall, in 3 out of 4 biopsies infiltrated byHPV16-specific T-cells, we were able to detect HPV16-specific CD8⁺ Tcells.Taken together, the population of immune cells migrating into the skinupon an intradermal challenge with HPV16 peptides comprisesHPV16-specific CD4⁺ Th1−, Th2− and CD8⁺ cytotoxic T cells. Thisinfiltration is paralleled by the appearance of circulatingHPV16-specific IFNγ-producing T-cells in the blood.

Discussion

Skin tests are commonly used as a simple assay for in vivo measurementof cell mediated immunity. We have validated the use of the skin testassay for the measurement of HPV 16 specific cellular immune responseagainst the early antigens E2, E6 and E7 in vivo by comparing theresults with that of parallel measurements of T cell reactivity by invitro assays.

The majority of positive skin reactions in patients with HPV relatedneoplasia appeared within 2-S days after administration of the skin testsimilar to what was observed in CIN patients by Hopfl et al. (Hopfl,2000). Also in the group of healthy volunteers early skin reactionsappeared between 4 to 12 days after intrademlal antigen challenge. Inthis latter group, known to display HPV16 specific type 1 T cellresponses in vitro (de Jong, 2002; Welters, 2003), the appearance of anearly skin reaction (within 13 days) was significantly associated withthe detection of IFNγ-producing HPV16-specific T cells at a frequency ofat least 1 per 20.000 PBMC (FIG. 2, p<0.001). The same cut-off criteriafor a positive reaction in the IFNγ ELispot assay are recommended byJeffries et al (Jeffries, 2006), who used mathematical tools to definethe appropriate cut-off of the ELISPOT in relation to Mantoux-tests. Thelow number of circulating memory T cells (FIG. 2) may explain why theskin reactions appear somewhat delayed compared to classical DTH tests.The T cells need to be boosted or reactivated and start to divide beforeenough cells are produced to cause a local inflammatory reaction: thepositive skin test. Indeed, at the time a positive skin reactionappears, a higher frequency of HPV16-specific Th1 responses can bedetected in the peripheral blood (FIG. 3).

In contrast to the healthy individuals, skin reactions in the patientpopulation were not associated with circulating HPV16 specific type 1 Tcells as measured by IFNγ ELispot, suggesting that HPV16 specific Tcells producing other cytokines than IFNγ infiltrated the skin test sitein patients. Historically it has been postulated that the Th1 cellinduce DTH responses, however, several studies have now shown that alsoTh2 cells infiltrating the skin test sites (Wang, 1999; Woodfolk, 2001).Similarly, this study shows that the positive skin test sites of healthyvolunteers contain both Th1 and Th2 type HPV16-specific T cells (FIGS. 4and 5). In addition, positive skin reactions may also be the result ofthe influx of non-specific T cells as became evident from two in depthstudies of positive skin test sites used to assay the specific immuneresponse following vaccination of patients with renal cell cancer ormelanoma (Bleumer, 2007). Also this study showed that a number ofpositive skin test sites from healthy subjects were infiltrated withT-cells that did not respond to the injected HPV16 antigens. So far, thereason for a-specific positive skin reactions remains unclear. However,based on these results and in view of our previous studies showing thatmost of the cervical carcinoma patients lack functional HPV-specificCD4+ T cell immunity (de Jong, 2004), we assume that the positive skinreactions in cancer patients are the result of either circulatingHPV-specific non-Th 1 cells or that of infiltrating T cells that arenot-specific for HPV16.

Unexpectedly, we observed the majority of skin reactions in healthyindividuals to appear 2 to 3 weeks after intradermal injection of theantigen. While, these late positive skin reactions were not correlatedwith detection of circulating HPV-specific CD4⁺ memory T cells inpre-challenge blood (FIG. 2) the immunological constitution of theseskin test sites are similar to that of classic DTH tests (Platt, 19S3;Poulter, 19S2) and comprised of HPV16-specific CD4+Th1- and Th2-cells aswell as HPV16-specific CDS+ T cells (FIGS. 4 and 5). We hypothesize thatthese reactions might be the result of T cell priming. This has alsobeen noted in 29% of patients whom underwent a 2-step tuberculin skintesting protocol and whom were only positive at the second test round(Akcay, 2003). In general, vaccine-induced T cell responses peak at 10to 14 days after vaccination and not at three weeks. However, one shouldbear in mind that in such protocol a higher antigen dose as well asstrong adjuvants are injected. It is therefore reasonable to assume thatthe T cell responses induced by intradermal challenge develop moreslowly and peak at a later period. Since the intra dermal peptidechallenge in healthy volunteers results in the induction of bothHPV16-specific CD4⁺ and CD8⁺ T cells it, therefore, should be consideredas a single, low dose vaccination.

The mam objective of this pilot study was to validate the use of theHPV16 specific skin test to detect type 1 immune responses in vivo. Inhealthy volunteers, a positive skin reaction within 13 days is indeedcorrelated with the presence of circulating IFNy-producing memory Tcells as detected by the IFNγ ELispot in vitro.

Importantly, we also observed discrepancies between the outcomesobtained by skin test and ELispot. In a number of cases HPV16-specificcirculating IFNy-producing T cells were detected in the post-challengeblood samples but without a concomitant skin reaction and vice versa(FIG. 3A), and this may be considered as a false negative or falsepositive result. In order to fully understand the impact of this on theinterpretation of the detection of type 1 immunity against HPV, we havebegun a field trial in a large group of patients and healthy volunteersin Indonesia.

TABLE 1 Patient characteristics pa- age diag- grade/ time tient (yrs)nosis stage Treatment (months)^(c) HPV 1 72 CxCa IB^(b) radicalhysterectomy 9 16 2 57 CxCa IA radical hysterectomy 18 16 3 57 CxCa IIBRadiotherapy 36 ? 4 50 CxCa IB radical hysterectomy 6 16 5 44 CxCa IAHysterectomy 36 16 6 53 CxCa IIB chemoradiation 36 16neg 7 34 CxCa IBradical hysterectomy 48 ? 8 44 CxCa IB radical hysterectomy 7 16 9 43CxCa IB radical hysterectomy 39 16 10 32 CxCa IA radical hysterectomy 20? 11 58 CxCa IB radical hysterectomy 74 18 12 44 CxCa IIA radicalhysterectomy 10 16 13 28 CIN I LEEP^(c) 0 neg 14 29 CIN III LEEP 2 ? 1542 CIN II LEEP 3 ? 16 49 CIN III LEEP 60 18 17 44 CIN III LEEP 12 16^(a)Time of treatment before skin tests were performed ^(b)cervicalcancer stage according to FIGO ^(c)Loop electrosurgical excisionprocedure

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1. A method for treatment of an HPV-induced intraepithelial neoplasia orcancer selected from a Human Papilloma Virus (HPV) infection, an HPVassociated malignancy, a Cervical Intra-epithelial Neoplasia (CIN), avulva Intra-epithelial Neoplasia (VIN), an Anal Intra-epithelialNeoplasia (AIN), a Vaginal Intra-epithelial Neoplasia (VAIN), a PenileIntra-epithelial Neoplasia (PIN), a cervical cancer, a vulva cancer, ananal cancer, a vaginal cancer, a head and neck cancer or a penilecancer, in a human in need thereof, the method comprising administeringintradermally to the human a composition comprising a pool of thefollowing peptides: (i) a peptide comprising SEQ ID NO: 7 and a peptidecomprising SEQ ID NO: 8; (ii) a peptide comprising SEQ ID NO: 9 and apeptide comprising SEQ ID NO: 10; (iii) a peptide comprising SEQ ID NO:11 and a peptide comprising SEQ ID NO: 12; (iv) a peptide comprising SEQID NO: 13, a peptide comprising SEQ ID NO: 14, and a peptide comprisingSEQ ID NO: 15; (v) a peptide comprising SEQ ID NO: 16 and a peptidecomprising SEQ ID NO: 17; (vi) a peptide comprising SEQ ID NO: 18 and apeptide comprising SEQ ID NO: 19; (vii) a peptide comprising SEQ ID NO:20 and a peptide comprising SEQ ID NO: 21; and/or, (vii) a combinationthereof, wherein the peptides are of 22-45 contiguous amino acids ofHPV-E2, -E6 or -E7 proteins and are administered in an amount suitableto elicit an antigen specific systemic cellular immune response withrespect to the peptides, wherein the composition does not comprise apharmaceutically acceptable adjuvant that has a depot function, andwherein the administering treats the HPV-induced intraepithelialneoplasia or cancer.
 2. The method according to claim 1, wherein thepeptides are of 22-40, 22-35, 22, 25, 28, 32 or 35 amino acids inlength.
 3. The method according to claim 1, wherein the compositionfurther comprises a contiguous amino acid sequence from HPV-E7 protein.4. The method according to claim 1, wherein the composition is anaqueous solution.
 5. The method according to claim 3, wherein thecontiguous amino acid sequence from the HPV-E7 protein comprises any oneof SEQ ID NOS: 20-23.
 6. The method according to claim 1, wherein thecomposition is not formulated as an oil-water based emulsion.
 7. Themethod according to claim 6, wherein the composition is not formulatedas a water-in-oil emulsion.
 8. The method according to claim 1, whereinthe composition consists of a pool of peptides as defined in claim 1,optionally further peptides selected from the group comprising SEQ IDNO: 7-23, and an inert pharmaceutically acceptable carrier.
 9. Themethod according to claim 1, wherein the composition comprises a TLRligand.
 10. The method according to claim 9, wherein the TLR ligand is aTLR 3, 4, 7, 8 and/or 9 ligand.
 11. The method according to claim 1,wherein the composition is intradermally administered directly at thesite of the lesion.
 12. The method according to claim 1, furthercomprising a second composition comprising a peptide derived fromHPV-E2, -E6 and/or -E7 protein, wherein the second composition isadministered subcutaneously.
 13. The method according to claim 1,wherein the antigen specific systemic immune response comprisescirculation of antigen specific T cells through the secondary lymphsystem of the subject.
 14. The method according to claim 1, wherein theantigen specific systemic immune response comprises circulation ofantigen specific T cells in the peripheral blood of the subject.
 15. Themethod according to claim 1, wherein the composition comprises anadditional peptide comprising any of the sequences of SEQ ID NO: 7-23.16. The method according to claim 15, wherein the composition is anaqueous solution.
 17. The method according to claim 3, wherein thecomposition is an aqueous solution.
 18. The method according to claim 1,wherein the composition comprises contiguous amino acid sequences fromeach of HPV-E2, -E6 and -E7 proteins.
 19. The method according to claim1, for treatment of an HPV-induced intraepithelial neoplasia or cancerselected from, a Cervical Intra-epithelial Neoplasia (CIN), a vulvaIntra-epithelial Neoplasia (VIN), an Anal Intra-epithelial Neoplasia(AIN), a cervical cancer, or a head and neck cancer.
 20. The methodaccording to claim 19, for treatment of an HPV-induced intraepithelialneoplasia or cancer selected from a Cervical Intra-epithelial Neoplasia(CIN), a vulva Intra-epithelial Neoplasia (VIN) or an AnalIntra-epithelial Neoplasia (AIN).